Development of Novel Phosphonodifluoromethyl-Containing Phosphotyrosine Mimetics and a First-In-Class, Potent, Selective, and Bioavailable Inhibitor of Human CDC14 Phosphatases.

Together with protein tyrosine kinases, protein tyrosine phosphatases (PTPs) control protein tyrosine phosphorylation and regulate numerous cellular functions. Dysregulated PTP activity is associated with the onset of multiple human diseases. Nevertheless, understanding of the physiological function and disease biology of most PTPs remains limited, largely due to the lack of PTP-specific chemical probes. In this study, starting from a well-known nonhydrolyzable phosphotyrosine (pTyr) mimetic, phosphonodifluoromethyl phenylalanine (F2Pmp), we synthesized 7 novel phosphonodifluoromethyl-containing bicyclic/tricyclic aryl derivatives with improved cell permeability and potency toward various PTPs. Furthermore, with fragment- and structure-based design strategies, we advanced compound 9 to compound 15, a first-in-class, potent, selective, and bioavailable inhibitor of human CDC14A and B phosphatases. This study demonstrates the applicability of the fragment-based design strategy in creating potent, selective, and bioavailable PTP inhibitors and provides a valuable probe for interrogating the biological roles of hCDC14 phosphatases and assessing their potential for therapeutic interventions.

Interactions of ubiquitin and CHMP5 with the V domain of HD-PTP reveals role for regulation of Vps4 ATPase.

The family of Bro1 proteins coordinates the activity of the Endosomal Sorting Complexes Required for Transport (ESCRTs) to mediate a number of membrane remodeling events. These events culminate in membrane scission catalyzed by ESCRT-III, whose polymerization and disassembly is controlled by the AAA-ATPase, Vps4. Bro1-family members Alix and HD-PTP as well as yeast Bro1 have central "V" domains that noncovalently bind Ub and connect ubiquitinated proteins to ESCRT-driven functions such as the incorporation of ubiquitinated membrane proteins into intralumenal vesicles of multivesicular bodies. Recently, it was discovered that the V domain of yeast Bro1 binds the MIT domain of Vps4 to stimulate its ATPase activity. Here we determine the structural basis for how the V domain of human HD-PTP binds ubiquitin. The HD-PTP V domain also binds the MIT domain of Vps4, and ubiquitin binding to the HD-PTP V domain enhances its ability to stimulate Vps4 ATPase activity. Additionally, we found that V domains of both HD-PTP and Bro1 bind CHMP5 and Vps60, respectively, providing another potential molecular mechanism to alter Vps4 activity. These data support a model whereby contacts between ubiquitin, ESCRT-III, and Vps4 by V domains of the Bro1 family may coordinate late events in ESCRT-driven membrane remodeling events.

Generation and characterization of a laforin nanobody inhibitor.

OBJECTIVES: Mutations in the gene encoding the glycogen phosphatase laforin result in the fatal childhood dementia Lafora disease (LD). A cellular hallmark of LD is cytoplasmic, hyper-phosphorylated, glycogen-like aggregates called Lafora bodies (LBs) that form in nearly all tissues and drive disease progression. Additional tools are needed to define the cellular function of laforin, understand the pathological role of laforin in LD, and determine the role of glycogen phosphate in glycogen metabolism. In this work, we present the generation and characterization of laforin nanobodies, with one being a laforin inhibitor. DESIGN AND METHODS: We identify multiple classes of specific laforin-binding nanobodies and determine their binding epitopes using hydrogen deuterium exchange (HDX) mass spectrometry. Using para-nitrophenyl phosphate (pNPP) and a malachite gold-based assay specific for glucan phosphatase activity, we assess the inhibitory effect of one nanobody on laforin's catalytic activity. RESULTS: Six families of laforin nanobodies are characterized and their epitopes mapped. One nanobody is identified and characterized that serves as an inhibitor of laforin's phosphatase activity. CONCLUSIONS: The six generated and characterized laforin nanobodies, with one being a laforin inhibitor, are an important set of tools that open new avenues to define unresolved glycogen metabolism questions.

Molecular dynamics simulation studies on the specific regulation of PTPN18 to the HER2 phospho-peptides.

The specific regulation of PTPN18 protein to three HER2 phospho-peptides has been studied by molecular dynamics simulations and free energy calculations. The results revealed that the three HER2 phospho-peptides binding to the PTPN18 catalytic domain is energetically favorable due to substrate specificity of PTPN18, and moreover, the PTPN18 protein have significantly higher affinity to pY1248 peptide (-45.22 kcal/mol) than that of pY1112 (-25.3 kcal/mol) and pY1196 (-31.86 kcal/mol) peptides. Further, the binding of HER2 phospho-peptides to PTPN18 have also caused the closure of WPD-loop with the decrease of the centroid distances between the P-loop and the WPD loop. The WPD-loop closure of PTPN18 relates directly to the new hydrogen bond and hydrophobic interaction formations between the residues Tyr62, Asp64, Val65, Ala231, Arg235, and Ala273 in PTPN18 and Tyr(PO3) in the HER2 phospho-peptides, which suggests that these key residues would contribute to the specific regulation of PTPN18 to the substrates. The correlation analysis revealed the allosteric communication networks from the pY binding loop to the WPD loop through the structural change and the residue interactions in PTPN18. These results will be helpful to understand the specific regulation through the allosteric communication network in the PTPN18 catalytic domain.

Molecular Analysis of the Interaction between Human PTPN21 and the Oncoprotein E7 from Human Papillomavirus Genotype 18.

Human papillomaviruses (HPVs) cause cellular hyperproliferation-associated abnormalities including cervical cancer. The HPV genome encodes two major viral oncoproteins, E6 and E7, which recruit various host proteins by direct interaction for proteasomal degradation. Recently, we reported the structure of HPV18 E7 conserved region 3 (CR3) bound to the protein tyrosine phosphatase (PTP) domain of PTPN14, a well-defined tumor suppressor, and found that this intermolecular interaction plays a key role in E7-driven transformation and tumorigenesis. In this study, we carried out a molecular analysis of the interaction between CR3 of HPV18 E7 and the PTP domain of PTPN21, a PTP protein that shares high sequence homology with PTPN14 but is putatively oncogenic rather than tumor-suppressive. Through the combined use of biochemical tools, we verified that HPV18 E7 and PTPN21 form a 2:2 complex, with a dissociation constant of 5 nM and a nearly identical binding manner with the HPV18 E7 and PTPN14 complex. Nevertheless, despite the structural similarities, the biological consequences of the E7 interaction were found to differ between the two PTP proteins. Unlike PTPN14, PTPN21 did not appear to be subjected to proteasomal degradation in HPV18-positive HeLa cervical cancer cells. Moreover, knockdown of PTPN21 led to retardation of the migration/invasion of HeLa cells and HPV18 E7-expressing HaCaT keratinocytes, which reflects its protumor activity. In conclusion, the associations of the viral oncoprotein E7 with PTPN14 and PTPN21 are similar at the molecular level but play different physiological roles.

Inhibition of striatal-enriched protein tyrosine phosphatase by targeting computationally revealed cryptic pockets.

Cryptic pockets, which are not apparent in crystallographic structures, provide promising alternatives to traditional binding sites for drug development. However, identifying cryptic pockets is extremely challenging and the therapeutic potential of cryptic pockets remains unclear. Here, we reported the discovery of novel inhibitors for striatal-enriched protein tyrosine phosphatase (STEP), a potential drug target for multiple neuropsychiatric disorders, based on cryptic pocket detection. By combining the use of molecular dynamics simulations and fragment-centric topographical mapping, we identified transiently open cryptic pockets and identified 12 new STEP inhibition scaffolds through structure-based virtual screening. Site-directed mutagenesis verified the binding of ST3 with the predicted cryptic pockets. Moreover, the most potent and selective inhibitors could modulate the phosphorylation of both ERK1/2 and Pyk2 in PC12 cells.

PTPN21-CDSlong isoform inhibits the response of acute lymphoblastic leukemia cells to NK-mediated lysis via the KIR/HLA-I axis.

Protein tyrosine phosphatase non-receptor type 21 (PTPN21) is a member of the non-receptor tyrosine phosphatase family. We have found that PTPN21 is mutated in relapsed Philadelphia chromosome-negative acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation. PTPN21 consists of three types of isoforms according to the length of the protein encoded. However, the roles of different isoforms in leukemic cells have not been elucidated. In the study, PTPN21 isoform constitution in five ALL cell lines were identified by transcriptome polymerase chain reaction combined with Sanger sequencing, and the relationship between PTPN21 isoforms and sensitivity to natural killer (NK) cells mediated killing in ALL cell lines were further assessed by knock-out of different isoforms of PTPN21 using CRISPR-Cas9 technique. Subsequently, we explored the functional mechanisms through RNA sequencing and confirmatory testing. The results showed that there was no significant change when all PTPN21 isoforms were knocked out in ALL cells, but the sensitivity of NALM6 cells with PTPN21-CDSlong knock-out (NALM6-PTPN21lk ) to NK-mediated killing was significantly increased. Whole transcriptome sequencing and further validation testing showed that human leukocyte antigen class I (HLA-I) molecules were significantly decreased, accompanied by a significantly downregulated expression of antigen presenting-related chaperones in NALM6-PTPN21lk cells. Our results uncovered a previously unknown mechanism that PTPN21-CDSlong and CDSshort isoforms may play opposite roles in NK-mediated killing in ALL cells, and showed that the endogenous PTPN21-CDSlong isoform inhibited ALL cells to NK cell-mediated lysis by regulating the KIR-HLA-I axis.

Regulation of the autophagic PI3KC3 complex by laforin/malin E3-ubiquitin ligase, two proteins involved in Lafora disease.

Lafora progressive myoclonus epilepsy is a fatal rare neurodegenerative disorder characterized by the accumulation of insoluble abnormal glycogen deposits in the brain and peripheral tissues. Mutations in at least two genes are responsible for the disease: EPM2A, encoding the glucan phosphatase laforin, and EPM2B, encoding the RING-type E3-ubiquitin ligase malin. Both laforin and malin form a functional complex in which laforin recruits the substrates to be ubiquitinated by malin. We and others have described that, in cellular and animal models of this disease, there is an autophagy impairment which leads to the accumulation of dysfunctional mitochondria. In addition, we established that the autophagic defect occurred at the initial steps of autophagosome formation. In this work, we present evidence that in cellular models of the disease there is a decrease in the amount of phosphatidylinositol-3P. This is probably due to defective regulation of the autophagic PI3KC3 complex, in the absence of a functional laforin/malin complex. In fact, we demonstrate that the laforin/malin complex interacts physically and co-localizes intracellularly with core components of the PI3KC3 complex (Beclin1, Vps34 and Vps15), and that this interaction is specific and results in the polyubiquitination of these proteins. In addition, the laforin/malin complex is also able to polyubiquitinate ATG14L and UVRAG. Finally, we show that overexpression of the laforin/malin complex increases PI3KC3 activity. All these results suggest a new role of the laforin/malin complex in the activation of autophagy via regulation of the PI3KC3 complex and explain the defect in autophagy described in Lafora disease.

The ESCRT-II Subunit EAP20/VPS25 and the Bro1 Domain Proteins HD-PTP and BROX Are Individually Dispensable for Herpes Simplex Virus 1 Replication.

Capsid envelopment during assembly of the neurotropic herpesviruses herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV) in the infected cell cytoplasm is thought to involve the late-acting cellular ESCRT (endosomal sorting complex required for transport) components ESCRT-III and VPS4 (vacuolar protein sorting 4). However, HSV-1, unlike members of many other families of enveloped viruses, does not appear to require the ESCRT-I subunit TSG101 or the Bro1 domain-containing protein ALIX (Alg-2-interacting protein X) to recruit and activate ESCRT-III. Alternative cellular factors that are known to be capable of regulating ESCRT-III function include the ESCRT-II complex and other members of the Bro1 family. We therefore used small interfering RNA (siRNA) to knock down the essential ESCRT-II subunit EAP20/VPS25 (ELL-associated protein 20/vacuolar protein sorting 25) and the Bro1 proteins HD-PTP (His domain-containing protein tyrosine phosphatase) and BROX (Bro1 domain and CAAX motif containing). We demonstrated reductions in levels of the targeted proteins by Western blotting and used quantitative microscopic assays to confirm loss of ESCRT-II and HD-PTP function. We found that in single-step replication experiments, the final yields of HSV-1 were unchanged following loss of EAP20, HD-PTP, or BROX.IMPORTANCE HSV-1 is a pathogen of the human nervous system that uses its own virus-encoded proteins and the normal cellular ESCRT machinery to drive the construction of its envelope. How HSV-1 structural proteins interact with ESCRT components and which subsets of cellular ESCRT proteins are utilized by the virus remain largely unknown. Here, we demonstrate that an essential component of the ESCRT-II complex and two ESCRT-associated Bro1 proteins are dispensable for HSV-1 replication.

Structural basis for recognition of the tumor suppressor protein PTPN14 by the oncoprotein E7 of human papillomavirus.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7's ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis.

Three novel MTM1 pathogenic variants identified in Japanese patients with X-linked myotubular myopathy.

BACKGROUND: X-linked myotubular myopathy (XLMTM) is a form of the severest congenital muscle diseases characterized by marked muscle weakness, hypotonia, and feeding and breathing difficulties in male infants. It is caused by mutations in the myotubularin gene (MTM1). METHODS: Evaluation of clinical history and examination of muscle pathology of three patients and comprehensive genome analysis on our original targeted gene panel system for muscular diseases. RESULTS: We report three patients, each of whom presents distinct muscle pathological features. The three patients have novel hemizygous MTM1 variants, including c.527A>G (p.Gln176Arg), c.595C>G (p.Pro199Ala), or c.688T>C (p.Trp230Arg). CONCLUSIONS: All variants were assessed as "Class 4 (likely pathogenic)" on the basis of the guideline of American College of Medical Genetics and Genomics. These distinct pathological features among the patients with variants in the second cluster of PTP domain in MTM1 provides an insight into microheterogeneities in disease phenotypes in XLMTM.

Structure and functions of His domain protein tyrosine phosphatase in receptor trafficking and cancer 1.

Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.

Allosteric Activation of Striatal-Enriched Protein Tyrosine Phosphatase (STEP, PTPN5) by a Fragment-like Molecule.

Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and 15N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.

Oligomerization and carbohydrate binding of glucan phosphatases.

Glucan phosphatases are a unique subset of the phosphatase family that bind to and dephosphorylate carbohydrate substrates. Family members are found in diverse organisms ranging from single-cell red algae to humans. The nature of their functional oligomerization has been a source of considerable debate. We demonstrate that the human laforin protein behaves aberrantly when subjected to Size Exclusion Chromotography (SEC) analysis due to interaction with the carbohydrate-based matrix. This interaction complicates the analysis of laforin human disease mutations. Herein, we show that SEC with Multi-Angle static Light Scattering (SEC-MALS) provides a method to robustly define the oligomerization state of laforin and laforin variants. We further analyzed glucan phosphatases from photosynthetic organisms to define if this interaction was characteristic of all glucan phosphatases. Starch EXcess-four (SEX4) from green plants was found to lack significant interaction with the matrix and instead exists as a monomer. Conversely, Cm-laforin, from red algae, exists as a monomer in solution while still exhibiting significant interaction with the matrix. These data demonstrate a range of oligomerization behaviors of members of the glucan phosphatase family, and establish SEC-MALS as a robust methodology to quantify and compare oligomerization states between different proteins and protein variants in this family.

The unique evolution of the carbohydrate-binding module CBM20 in laforin.

Laforin catalyses glycogen dephosphorylation. Mutations in its gene result in Lafora disease, a fatal progressive myoclonus epilepsy, the hallmark being water-insoluble, hyperphosphorylated carbohydrate inclusions called Lafora bodies. Human laforin consists of an N-terminal carbohydrate-binding module (CBM) from family CBM20 and a C-terminal dual-specificity phosphatase domain. Laforin is conserved in all vertebrates, some basal metazoans and a small group of protozoans. The present in silico study defines the evolutionary relationships among the CBM20s of laforin with an emphasis on newly identified laforin orthologues. The study reveals putative laforin orthologues in Trichinella, a parasitic nematode, and identifies two sequence inserts in the CBM20 of laforin from parasitic coccidia. Finally, we identify that the putative laforin orthologues from some protozoa and algae possess more than one CBM20.

The MTM1-UBQLN2-HSP complex mediates degradation of misfolded intermediate filaments in skeletal muscle.

The ubiquitin proteasome system and autophagy are major protein turnover mechanisms in muscle cells, which ensure stemness and muscle fibre maintenance. Muscle cells contain a high proportion of cytoskeletal proteins, which are prone to misfolding and aggregation; pathological processes that are observed in several neuromuscular diseases called proteinopathies. Despite advances in deciphering the mechanisms underlying misfolding and aggregation, little is known about how muscle cells manage cytoskeletal degradation. Here, we describe a process by which muscle cells degrade the misfolded intermediate filament proteins desmin and vimentin by the proteasome. This relies on the MTM1-UBQLN2 complex to recognize and guide these misfolded proteins to the proteasome and occurs prior to aggregate formation. Thus, our data highlight a safeguarding function of the MTM1-UBQLN2 complex that ensures cytoskeletal integrity to avoid proteotoxic aggregate formation.

Whole-Exome Sequencing Identifies a Novel Homozygous Frameshift Mutation in the MTMR2 Gene as a Causative Mutation in a Patient with Charcot-Marie-Tooth Disease Type 4B1.

Charcot-Marie-Tooth (CMT) disease refers to a heterogeneous group of axonal and demyelinating polyneuropathies, characterized by chronic motor and sensory dysfunction. CMT is the most common genetic cause of neuropathy. The present study aimed to identify the gene mutation responsible for CMT in Ashkenazi Jew (AJ) patient. Genomic DNA was extracted from whole blood leukocytes of affected family and normal subject. Whole-exome sequencing was performed using the Illumina HiSeq2500. The DNA region containing the identified mutation was amplified by PCR and sequenced using dye-terminator chemistry and the forward primer. Physical examination of the patient revealed weakness and atrophy of the lower extremity muscles and Pes cavus foot deformity. Whole-exome sequencing indicated that the patient is homozygous for a novel frameshift mutation (c.1877_1878insAGAG, p.Arg630fs) in the myotubularin-related protein-2 gene (MTMR2), which resulted in an erroneous C-terminal sequence and extension by 15 amino acids. Patients' parents are healthy, and DNA sequencing analysis indicated that both are heterozygotes to the described mutation. The clinical feature of the patient may indicate a complete co-segregation of the p.Arg630fs mutation in MTMR2 gene with the CMT type 4B1 phenotype. Further studies are needed in order to estimate the prevalence of this mutation among AJ.

Mutations of PTPN23 in developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEE) are a heterogeneous group of neurodevelopmental disorders with poor prognosis. Recent discoveries have greatly expanded the repertoire of genes that are mutated in epileptic encephalopathies and DEE, often in a de novo fashion, but in many patients, the disease remains molecularly uncharacterized. Here, we describe a new form of DEE in patients with likely deleterious biallelic variants in PTPN23. The phenotype is characterized by early onset drug-resistant epilepsy, severe and global developmental delay, microcephaly, and sometimes premature death. PTPN23 encodes a tyrosine phosphatase with strong brain expression, and its knockout in mouse is embryonically lethal. Structural modeling supports a deleterious effect of the identified alleles. Our data suggest that PTPN23 mutations cause a rare severe form of autosomal-recessive DEE in humans, a finding that requires confirmation.

X-ray Characterization and Structure-Based Optimization of Striatal-Enriched Protein Tyrosine Phosphatase Inhibitors.

Excessive activity of striatal-enriched protein tyrosine phosphatase (STEP) in the brain has been detected in numerous neuropsychiatric disorders including Alzheimer's disease. Notably, knockdown of STEP in an Alzheimer mouse model effected an increase in the phosphorylation levels of downstream STEP substrates and a significant reversal in the observed cognitive and memory deficits. These data point to the promising potential of STEP as a target for drug discovery in Alzheimer's treatment. We previously reported a substrate-based approach to the development of low molecular weight STEP inhibitors with Ki values as low as 7.8 µM. Herein, we disclose the first X-ray crystal structures of inhibitors bound to STEP and the surprising finding that they occupy noncoincident binding sites. Moreover, we utilize this structural information to optimize the inhibitor structure to achieve a Ki of 110 nM, with 15-60-fold selectivity across a series of phosphatases.

The open architecture of HD-PTP phosphatase provides new insights into the mechanism of regulation of ESCRT function.

HD-PTP is a tumour suppressor phosphatase that controls endocytosis, down-regulation of mitogenic receptors and cell migration. Central to its role is the specific recruitment of critical endosomal sorting complexes required for transport (ESCRTs). However, the molecular mechanisms that enable HD-PTP to regulate ESCRT function are unknown. We have characterised the molecular architecture of the entire ESCRT binding region of HD-PTP using small angle X-ray scattering and hydrodynamic analyses. We show that HD-PTP adopts an open and extended conformation, optimal for concomitant interactions with multiple ESCRTs, which contrasts with the compact conformation of the related ESCRT regulator Alix. We demonstrate that the HD-PTP open conformation is functionally competent for binding cellular protein partners. Our analyses rationalise the functional cooperation of HD-PTP with ESCRT-0, ESCRT-I and ESCRT-III and support a model for regulation of ESCRT function by displacement of ESCRT subunits, which is crucial in determining the fate of ubiquitinated cargo.